Malate thiokinase catalyzes an ATP dependent thiol ester formation between Coenzyme A and a number of dicarboxylic acids. The reaction is analogous to the well known succinate thiokinase reaction. Upon purification of malate thiokinase using 3' -5' AMP coupled to Sepharose 4B, two active forms of the enzyme have been observed as judged by electrophoresis on polyacrylamide gels. One form has been identified as the native form of the enzyme, mol wt approximately 180,000 and composed of 4 alpha and 4 beta subunits. The other active form of the enzyme is produced by an interaction of succinyl CoA with the enzyme, has a molecular weight of approximately 100,000 and appears to be composed of 2 alpha and 2 beta subunits. The S-form of the enzyme can be converted back to the N-form by reaction with ATP or with ADP plus Pi. Using (14C)-succinyl CoA and succinyl-(3H) CoA preliminary experiments indicate labeling of the alpha-subunit by the succinyl moiety with the loss of Coenzyme A. These results suggest the formation of a succinyl-enzyme intermediate in the malate thiokinase reaction.